Structural Design of the Active Site for Covalent Attachment of the Heme to the Protein Matrix: Studies on a Thermostable Cytochrome P450

Biochemistry ◽  
2011 ◽  
Vol 50 (6) ◽  
pp. 1042-1052 ◽  
Author(s):  
Sandeep Goyal ◽  
Megha S. Deshpande ◽  
Shyamalava Mazumdar
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Structural Design ◽  
Covalent Attachment ◽  
Protein Matrix

The Molecular and Enzyme Kinetic Basis for Altered Activity of Three Cytochrome P450 2C19 Variants Found in the Chinese Population

2020 ◽  
Vol 13 (3) ◽  
pp. 233-244
Author(s):  
Amelia Nathania Dong ◽  
Nafees Ahemad ◽  
Yan Pan ◽  
Uma Devi Palanisamy ◽  
Beow Chin Yiap ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Molecular Docking ◽  
Active Site ◽  
Chinese Population ◽  
Accessible Surface Area ◽  
Solvent Accessible Surface Area ◽  
In Vitro Assays ◽  
Access Channel ◽  
Cytochrome P450 2C19

Background: There is a large inter-individual variation in cytochrome P450 2C19 (CYP2C19) activity. The variability can be caused by the genetic polymorphism of CYP2C19 gene. This study aimed to investigate the molecular and kinetics basis for activity changes in three alleles including CYP2C19*23, CYP2C19*24 and CYP2C19*25found in the Chinese population. Methods: The three variants expressed by bacteria were investigated using substrate (omeprazole and 3- cyano-7-ethoxycoumarin[CEC]) and inhibitor (ketoconazole, fluoxetine, sertraline and loratadine) probes in enzyme assays along with molecular docking. Results: All alleles exhibited very low enzyme activity and affinity towards omeprazole and CEC (6.1% or less in intrinsic clearance). The inhibition studies with the four inhibitors, however, suggested that mutations in different variants have a tendency to cause enhanced binding (reduced IC50 values). The enhanced binding could partially be explained by the lower polar solvent accessible surface area of the inhibitors relative to the substrates. Molecular docking indicated that G91R, R335Q and F448L, the unique mutations in the alleles, have caused slight alteration in the substrate access channel morphology and a more compact active site cavity hence affecting ligand access and binding. It is likely that these structural alterations in CYP2C19 proteins have caused ligand-specific alteration in catalytic and inhibitory specificities as observed in the in vitro assays. Conclusion: This study indicates that CYP2C19 variant selectivity for ligands was not solely governed by mutation-induced modifications in the active site architecture, but the intrinsic properties of the probe compounds also played a vital role.

scholarly journals Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

2006 ◽  
Vol 455 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Cynthia E. Hernandez ◽  
Santosh Kumar ◽  
Hong Liu ◽  
James R. Halpert
Keyword(s):  
Cytochrome P450 ◽  
Crystal Structures ◽  
Active Site ◽  
Active Site Residues ◽  
Substrate Metabolism ◽  
Cytochrome P450 2B4

Substrate Specificity for the Epoxidation of Terpenoids and Active Site Topology of House Fly Cytochrome P450 6A1

1997 ◽  
Vol 10 (2) ◽  
pp. 156-164 ◽  
Author(s):  
John F. Andersen ◽  
Jennifer K. Walding ◽  
Philip H. Evans ◽  
William S. Bowers ◽  
René Feyereisen
Keyword(s):  
Cytochrome P450 ◽  
Substrate Specificity ◽  
Active Site ◽  
House Fly

scholarly journals Specificity and mechanism of carbohydrate demethylation by cytochrome P450 monooxygenases

2018 ◽  
Vol 475 (23) ◽  
pp. 3875-3886 ◽  
Author(s):  
Craig S. Robb ◽  
Lukas Reisky ◽  
Uwe T. Bornscheuer ◽  
Jan-Hendrik Hehemann
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Substrate Recognition ◽  
High Energy ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
High Energy Barrier ◽  
Molecular Determinants ◽  
Cytochrome P450 Family ◽  
Carbohydrate Ligand

Degradation of carbohydrates by bacteria represents a key step in energy metabolism that can be inhibited by methylated sugars. Removal of methyl groups, which is critical for further processing, poses a biocatalytic challenge because enzymes need to overcome a high energy barrier. Our structural and computational analysis revealed how a member of the cytochrome P450 family evolved to oxidize a carbohydrate ligand. Using structural biology, we ascertained the molecular determinants of substrate specificity and revealed a highly specialized active site complementary to the substrate chemistry. Invariance of the residues involved in substrate recognition across the subfamily suggests that they are critical for enzyme function and when mutated, the enzyme lost substrate recognition. The structure of a carbohydrate-active P450 adds mechanistic insight into monooxygenase action on a methylated monosaccharide and reveals the broad conservation of the active site machinery across the subfamily.

Differential Contribution of Active Site Residues in Substrate Recognition Sites 1 and 5 to Cytochrome P450 2C8 Substrate Selectivity and Regioselectivity†

Biochemistry ◽  
2004 ◽  
Vol 43 (24) ◽  
pp. 7834-7842 ◽  
Author(s):  
Oranun Kerdpin ◽  
David J. Elliot ◽  
Sanford L. Boye ◽  
Donald J. Birkett ◽  
Krongtong Yoovathaworn ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Substrate Recognition ◽  
Substrate Selectivity ◽  
Active Site Residues ◽  
Recognition Sites ◽  
Cytochrome P450 2C8 ◽  
Differential Contribution

Covalent Attachment of the Heme Prosthetic Group in the CYP4F Cytochrome P450 Family†

Biochemistry ◽  
2002 ◽  
Vol 41 (18) ◽  
pp. 5931-5937 ◽  
Author(s):  
Laurie A. LeBrun ◽  
Fengyun Xu ◽  
Deanna L. Kroetz ◽  
Paul R. Ortiz de Montellano
Keyword(s):  
Cytochrome P450 ◽  
Covalent Attachment ◽  
Prosthetic Group ◽  
Heme Prosthetic Group ◽  
Cytochrome P450 Family

scholarly journals Active-site differences between substrate-free and ritonavir-bound cytochrome P450 (CYP) 3A5 reveal plasticity differences between CYP3A5 and CYP3A4

2019 ◽  
Vol 294 (20) ◽  
pp. 8015-8022 ◽  
Author(s):  
Mei-Hui Hsu ◽  
Eric F. Johnson
Keyword(s):  
Cytochrome P450 ◽  
Drug Metabolism ◽  
Active Site ◽  
Drug Efficacy ◽  
Structural Features ◽  
Crystallographic Structure ◽  
Access Channel ◽  
Active Site Cavity ◽  
Human Adults ◽  
Related Enzyme

Cytochrome P450 (CYP) 3A4 is a major contributor to hepatic drug and xenobiotic metabolism in human adults. The related enzyme CYP3A5 is also expressed in adult liver and has broader age and tissue distributions. However, CYP3A5 expression is low in most Caucasians because of the prevalence of an allele that leads to an incorrectly spliced mRNA and premature termination of translation. When expressed, CYP3A5 expands metabolic capabilities and can augment CYP3A4-mediated drug metabolism, thereby reducing drug efficacy and potentially requiring dose adjustments. The extensive role of CYP3A4 in drug metabolism reflects in part the plasticity of the substrate-free enzyme to enlarge its active site and accommodate very large substrates. We have previously shown that the structure of the CYP3A5–ritonavir complex differs substantially from that of the CYP3A4–ritonavir complex. To better understand whether these differences are conserved in other CYP3A5 structures and how they relate to differential plasticity, we determined the X-ray crystallographic structure of the CYP3A5 substrate-free complex to 2.20 Å resolution. We observed that this structure exhibits a much larger active site than substrate-free CYP3A4 and displays an open substrate access channel. This reflected in part a lower trajectory of the helix F–F′ connector in CYP3A4 and more extensive π–CH interactions between phenylalanine residues forming the roof of the active-site cavity than in CYP3A5. Comparison with the CYP3A5–ritonavir complex confirmed conserved CYP3A5 structural features and indicated differences in plasticity between CYP3A4 and CYP3A5 that favor alternative ritonavir conformations.

scholarly journals A Kinetic Model for the Metabolic Interaction of Two Substrates at the Active Site of Cytochrome P450 3A4

2000 ◽  
Vol 276 (3) ◽  
pp. 2256-2262 ◽  
Author(s):  
Magang Shou ◽  
Renke Dai ◽  
Dan Cui ◽  
Kenneth R. Korzekwa ◽  
Thomas A. Baillie ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Kinetic Model ◽  
Active Site ◽  
Cytochrome P450 3A4 ◽  
Metabolic Interaction

scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

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